Pharmaceutical compositions containing a pde4 inhibitor and a p13 delta or dual p13 delta-gamma kinase inhibitor

ABSTRACT

This present invention relates to a method of treating a autoimmune, respiratory and/or inflammatory disease or condition (e.g., psoriasis, rheumatoid arthritis, asthma, COPD). The method comprises administering a PI3K Delta inhibitor or a dual PI3K Delta-Gamma inhibitor and a PDE4 inhibitor. The present invention also relates to pharmaceutical compositions containing a PI3K Delta or dual PI3K Delta-Gamma inhibitor and a PDE4 inhibitor.

The present application claims the benefit of Indian Patent ApplicationNos. 2762/CHE/2012, filed Nov. 8, 2012, and 4688/CHE/2012, filed Nov. 8,2012, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method of treating an autoimmune,respiratory and/or inflammatory disease or condition (such as psoriasis,rheumatoid arthritis, asthma, and COPD) by administering a PI3K Deltainhibitor or a dual PI3K Delta-Gamma inhibitor and a PDE4 inhibitor. Thepresent invention also relates to pharmaceutical compositions containinga PI3K Delta or dual PI3K Delta-Gamma inhibitor and a PDE4 inhibitor.

BACKGROUND OF THE INVENTION

Autoimmune, respiratory and inflammatory diseases such as rheumatoidarthritis (RA), psoriasis, systemic lupus erythematosus (SLE), chronicobstructive pulmonary disease (COPD), and asthma are chronic and oftenprogressive diseases associated with a dysregulated or overactive immunesystem. The causes and the drivers of these diseases remain ill-defined.They are characterized by complex cellular interactions between multipleinflammatory cells of the innate and adaptive immune system.Accordingly, the heterogeneity and complexity of the disease etiology ofthese conditions makes the search for new cellular targets challenging,as it is unclear who in the cellular infiltrate is a primary player ofthe pathology versus an “innocent” bystander.

Rheumatoid arthritis (RA) is a progressive, systemic autoimmune diseasecharacterized by chronic inflammation of multiple joints with associatedsystemic symptoms such as fatigue. This inflammation causes joint pain,stiffness and swelling, resulting in loss of joint function due todestruction of the bone and cartilage, often leading to progressivedisability. Patients with RA also have an increased likelihood ofdeveloping other systemic complications such as osteoporosis, anaemia,and other disorders affecting the lungs and skin. The disease alsoimpacts on the average life expectancy, shortening it by three to sevenyears.

There are a number of treatments available to manage RA. Some addressthe signs and symptoms of RA, others aim to modify the course of thedisease and positively impact the systemic effects of RA, such asfatigue and anaemia.

The current treatments include use of:

Biologics: These are genetically-engineered drugs that target specificcell surface markers or messenger substances in the immune system calledcytokines, which are produced by cells in order to regulate other cellsduring an inflammatory response. An example of a specific cytokinetargeted by biologics is tumor necrosis factor alpha (TNFα).

Traditional disease-modifying anti-rheumatic drugs (DMARDs): These arenon-specific immunosuppressive drugs, intended to combat the signs andsymptoms of RA as well as slowing down progressive joint destruction.These treatments are often used in combination with one another, or incombination with a biologic agent, to improve patient response.

Glucocorticoids (corticosteroids): These are anti-inflammatory drugsrelated to cortisol, a steroid produced naturally in the body, that workby countering inflammation. However, the side-effects ofglucocorticoids, which include hyperglycaemia, osteoporosis,hypertension, weight gain, cataracts, sleep problems, muscle loss, andsusceptibility to infections, limit their use.

Non-steroidal anti-inflammatory drugs (NSAIDs): These manage the signsand symptoms of RA, such as reducing pain, swelling, and inflammation,but do not alter the course of the disease or slow the progression ofjoint destruction.

There are also a number of RA therapies targeting other components ofthe immune system. These include biologic treatments targetingalternative cytokines such as interleukin-6 (IL-6) that help to reduceinflammation and the progression of RA in the joints and throughout thebody.

Asthma is the most common chronic disease among children and alsoaffects millions of adults. Some 235 million people worldwide sufferfrom this disease. The causes of asthma, however, are not wellunderstood.

Chronic obstructive pulmonary disease (COPD) is a highly prevalentcondition and a major cause of morbidity and mortality worldwide. As thedisease progresses, patients with COPD may become prone to frequentexacerbations, resulting in patient anxiety, worsening health status,lung function decline, and increase in mortality rate. These episodes ofworsening respiratory function lead to increases in health careutilization, hospital admissions and costs. Worse, frequentexacerbations are associated with a faster decline in lung function,thereby shortening life expectancy.

According to the recommendations of Global Initiative for ChronicObstructive Lung Disease (GOLD), the first line therapy for COPD arelong acting β-agonists, long acting muscarinic antagonist and inhalationcorticosteroids. However, these drugs reduce the symptoms andexacerbations associated with the disease, rather than targeting itsmolecular and cellular basis. Accordingly, there is still a need forfurther improvement of COPD therapy.

Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellularlipid kinases that phosphorylate the 3 position hydroxyl group of theinositol ring of phosphoinositide lipids (PIs) generating lipid secondmessengers. There are four claim I PI3K isoforms alpha, beta, delta, andgamma.

IC87114 (2-((6-amino-9H-purin-9-yl)methyl)-5-methyl-3-o-tolylquinazolin-4(3H)-one) is a specific PI3Kδinhibitor (Sadhu, J. Immunology, 1; 170(5):2647-2654, 2003;International Publication No. WO 2010/111432 and U.S. Publication Nos.2010/0249155 and 2010/0168139).

CAL-101 (Idelalisib), TGR-1202, AMG-319, and INCB040093 have beenreported as inhibitors of PI3Kδ and are under active clinicaldevelopment. IPI-145 (duvelisib) and CAL130 have been reported to asdual inhibitors of PI3K δ/γ, IPI-145 is under clinical investigation forcancer and asthma, and for RA in combination with methotrexate.

Phosphodiesterase-4 (PDE4) inhibition is one approach to the treatmentof COPD. Roflumilast, a new PDE4 inhibitor, reduces airway inflammationin COPD, as assessed with sputum neutrophil and eosinophil counts.However, roflumilast exhibits dose dependent toxicity which limits theuse of roflumilast at higher doses. Calverley, P, Rabe K, et. al,Roflumilast in Symptomatic Chronic Obstructive Pulmonary Disease: TwoRandomized Clinical Trials. The Lancet 2009; 374: 685-694 and Fabbri, L,Calverley, P, et. al, Roflumilast in Moderate to Severe ChronicObstructive Pulmonary Disease Treated with Longacting Bronchodilators:Two Randomized Clinical Trials. The Lancet 2009; 374: 695-703.

In addition, Celgene has shown positive results in two phase III studiesfor the PDE-4 inhibitor aprelimilast in the treatment of psoriaticarthritis.

Another PDE4 inhibitor is AN2728 from Anacor Pharmaceuticals which hascompleted phase II studies for atopic dermatitis. Recently, Chiesi Groupannounced the successful completion of its Phase I trial of CHF6001, aninhaled PDE4 inhibitor being developed for the treatment of inflammatoryrespiratory disorders, such as chronic obstructive pulmonary disease(COPD) and asthma.

PDE4 inhibitors, however, have a narrow therapeutic window as far asefficacy and toxicity are concerned.

Despite currently available intervention therapies, improved medicaltreatments for autoimmune disorders, such as RA and psoriasis, andrespiratory disorders, such as asthma and COPD, are still needed.

Accordingly, it is an objective of the present invention to provide newpharmaceutical compositions and methods for the treatment of respiratoryand/or inflammatory diseases and conditions having enhanced activity.

SUMMARY OF THE INVENTION

The present invention relates to a method of treating an autoimmune,respiratory and/or inflammatory disease or condition, such as rheumatoidarthritis (RA) or chronic obstructive pulmonary disease (COPD). Themethod involves administering a combination of (i) a PI3K Delta or adual PI3K Delta and Gamma inhibitor and (ii) a PDE4 inhibitor. It hasbeen surprisingly found that a PI3K Delta or dual PI3K Delta and Gammainhibitor acts synergistically with a PDE-4 inhibitor (i.e., thecombination exhibits an activity which is significantly greater than theactivity that would have been expected based on the individualactivities of each of the PI3K Delta or dual PI3K Delta and Gammainhibitor and the PDE-4 inhibitor alone). The PI3K Delta or a dual PI3KDelta and Gamma inhibitor and PDE4 inhibitor may be co-administered (forexample, by having both in a single dosage form or administeringseparate dosage forms simultaneously or sequentially). This combinationis particularly useful for treating asthma, allergic rhinitis,non-allergic rhinitis, RA, COPD and atopic dermatitis.

One embodiment is a method of treating an autoimmune, respiratory and/orinflammatory disease or condition (such as RA or COPD) comprisingadministering to a patient in need thereof a PI3K Delta inhibitor and aPDE4 inhibitor. Preferably, a therapeutically effective amount of thePI3K Delta inhibitor and PDE4 inhibitor are administered.

Another embodiment is a method of treating an autoimmune, respiratoryand/or inflammatory disease or condition (such as RA or COPD) comprisingadministering to a patient in need thereof a dual PI3K Delta and Gammainhibitor and a PDE4 inhibitor. Preferably, a therapeutically effectiveamount of the dual PI3K Delta and Gamma inhibitor and PDE4 inhibitor areadministered.

Yet another embodiment is a pharmaceutical composition comprising (i) aPI3K Delta or a dual PI3K Delta and Gamma inhibitor and (ii) a PDE4inhibitor. The pharmaceutical composition may be used for the treatmentof autoimmune, respiratory and inflammatory diseases and conditions,such as the treatment of RA and COPD.

In one embodiment, the pharmaceutical composition comprises a PI3K Deltainhibitor and a PDE4 inhibitor. In another embodiment, thepharmaceutical composition comprises a dual PI3K Delta and Gammainhibitor and a PDE4 inhibitor. In one preferred embodiment, thepharmaceutical composition includes a therapeutically effective amountof the PI3K Delta or dual PI3K Delta and Gamma inhibitor and PDE4inhibitor.

The methods and compositions described herein allow for the treatment ofautoimmune, respiratory and/or inflammatory diseases and conditions witha smaller amount of active agent, thereby allowing for costs savings,reduced side effects and allowing treatment to be continued for a longerperiod of time in a more efficient way.

In one embodiment, the PI3K Delta or dual PI3K Delta and Gamma inhibitoris a compound of formula (I):

or a tautomer thereof, N-oxide thereof, pharmaceutically acceptableester thereof, prodrug thereof, or pharmaceutically acceptable saltthereof, wherein

each occurrence of R is independently selected from hydrogen, halogen,—OR^(a), CN, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆ alkynyl,substituted or unsubstituted C₃₋₈ cycloalkyl, and substituted orunsubstituted heterocyclic group;

R¹ and R² may be the same or different and are independently selectedfrom hydrogen, halogen, and substituted or unsubstituted C₁₋₆ alkyl, orboth R¹ and R² directly bound to a common atom, may be joined to form anoxo group (═O) or a substituted or unsubstituted saturated orunsaturated 3-10 member ring (including the carbon atom to which R¹ andR² are bound), which may optionally include one or more heteroatomswhich may be the same or different and are selected from 0, NW and S;

Cy¹ is a monocyclic group selected from substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocyclic group, substitutedor unsubstituted aryl and substituted or unsubstituted heteroaryl;

Cy² is selected from a substituted or unsubstituted heterocyclic group,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl;

L₁ is absent or selected from —(CR^(a)R^(b))_(q)—, —O—, —S(═O)_(q)—,—NR^(a)— or —C(═Y)—; each occurrence of R^(a) and R^(b) may be the sameor different and are independently selected from hydrogen, halogen,hydroxy, cyano, substituted or unsubstituted (C₁₋₆)alkyl, —NR^(c)R^(d)(wherein R^(c) and R^(d) are independently hydrogen, halogen, hydroxy,cyano, substituted or unsubstituted (C₁₋₆)alkyl, and (C₁₋₆)alkoxy) and—OR^(c) (wherein W is substituted or unsubstituted (C₁₋₆)alkyl) or whenR^(a) and R^(b) are directly bound to a common atom, they may be joinedto form an oxo group (═O) or form a substituted or unsubstitutedsaturated or unsaturated 3-10 member ring (including the common atom towhich R^(a) and R^(b) are directly bound), which may optionally includeone or more heteroatoms which may be the same or different and areselected from O, NR^(d) (wherein R^(d) is hydrogen or substituted orunsubstituted (C₁₋₆)alkyl) or S;

Y is selected from O, S, and NR^(a);

n is 1, 2, 3 or 4; and

q is 0, 1 or 2.

In one embodiment, the compound of formula (I) is a compound of formula(II):

or a tautomer thereof, N-oxide thereof, pharmaceutically acceptableester thereof, prodrug thereof, or pharmaceutically acceptable saltthereof, wherein R¹, R², L₁, Cy¹ and Cy² are as described above forformula (I).

In further embodiments, the compound of formula (I) is selected from acompound of formulas (IA-I), (IA-II), (IA-HI) and (IA-IV):

or a tautomer thereof, N-oxide thereof, pharmaceutically acceptableester thereof, prodrug thereof, or pharmaceutically acceptable saltthereof, wherein

R, Cy¹, R¹, R², n and q are as defined above;

each occurrence of X is independently selected from CR³ or N; and

each occurrence of R³ is independently selected from hydrogen, hydroxy,halogen, carboxyl, cyano, nitro, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted arylalkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkylalkyl, substituted or unsubstituted cycloalkenylalkylsubstituted or unsubstituted cycloalkenyl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted heterocyclic ring, substituted heterocyclylalkyl ring,substituted or unsubstituted guanidine, —COOR^(x), —C(O)R^(x),—C(S)R^(x), —C(O)NR^(x)R^(y), —C(O)ONR^(x)R^(y), —NR^(y)R^(z),—NR^(x)CONR^(y)R^(z), —N(R^(x))SOR^(y), —N(R^(x))SO₂R^(y),—(═N—N(R^(x))R^(y)), —NR^(x)C(O)OR^(y), —NR^(x)R^(y), —NR^(x)C(O)R^(y)—,—NR^(x)C(S)R^(y), —NR^(x)C(S)NR^(y)R^(z), —SONR^(x)R^(y)—,—SO₂NR^(x)R^(y)—, —OR^(x), —OR^(x)C(O)NR^(y)R^(z), —OR^(x)C(O)OR^(y)—,—OC(O)R^(x), —OC(O)NR^(x)R^(y), —R^(x)NR^(y)C(O)R^(z), —R^(x)OR^(y),—R^(x)C(O)OR^(y), —R^(x)C(O)NR^(y)R^(z), —R^(x)C(O)R^(x),—R^(x)OC(O)R^(y), —SR^(x), —SOR^(x), —SO₂R^(x), and —ONO₂, whereinR^(x), R^(y) and R^(z) in each of the above groups can be hydrogen,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted aryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted cycloalkylalkyl, substituted or unsubstitutedcycloalkenyl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclicring, substituted or unsubstituted heterocyclylalkyl ring, orsubstituted or unsubstituted amino, or any two of R^(x), R^(y) and R^(z)may be joined to form a substituted or unsubstituted saturated orunsaturated 3-10 membered ring, which may optionally include heteroatomswhich may be the same or different and are selected from O, NR^(f)(wherein R^(f) is hydrogen or substituted or unsubstituted alkyl) or S.

In a preferred embodiment, the present invention relates to method oftreating an autoimmune, respiratory and/or inflammatory disease orcondition comprising administering a combination of a PI3K Delta or dualPI3K Delta and Gamma inhibitor and a PDE4 inhibitor, where the PI3KDelta or dual PI3K Delta and Gamma inhibitor is a compound of formula(I), (II), (IA-I), (IA-II), (IA-HI), or (IA-IV), as described above.

In another preferred embodiment, the present invention relates to apharmaceutical composition comprising a PI3K Delta or dual PI3K Deltaand Gamma inhibitor and a PDE4 inhibitor, where the PI3K Delta or dualPI3K Delta and Gamma inhibitor is a compound of formula (I), (II),(IA-I), (IA-II), (IA-HI), or (IA-IV), as described above.

In a preferred embodiment, the compound of formula (I) is selected from:

-   2-[(6-Amino-9H-purin-9-yl)    methyl]-6-bromo-3-phenyl-4H-chromen-4-one;-   6-Bromo-2-(morpholinomethyl)-3-phenyl-4H-chromen-4-one;-   6-Bromo-2-(morpholinomethyl)-3-phenyl-4H-chromen-4-one    hydrochloride;-   2-[(6-Amino-9H-purin-9-yl) methyl]-3-phenyl-4H-chromen-4-one;-   2-(Morpholinomethyl)-3-phenyl-4H-chromen-4-one;-   2-(Morpholinomethyl)-3-phenyl-4H-chromen-4-one hydrochloride;-   2-[(1H-Benzo[d]imidazol-1-yl)    methyl]-6-bromo-3-phenyl-4H-chromen-4-one;-   6-Bromo-2-[(4-methyl-1H-benzo[d]imidazol-1-yl)    methyl]-3-phenyl-4H-chromen-4-one;-   2-[(1H-benzo[d]imidazol-1-yl) methyl]-3-phenyl-4H-chromen-4-one;-   2-[(4-methyl-1H-benzo[d]imidazol-1-yl)    methyl]-3-phenyl-4H-chromen-4-one;-   2-[(6-Chloro-9H-purin-9-yl) methyl]-3-phenyl-4H-chromen-4-one;-   6-Bromo-2-[(6-chloro-9H-purin-9-yl)    methyl]-3-phenyl-4H-chromen-4-one;-   2-((9H-Purin-6-ylthio) methyl)-3-phenyl-4H-chromen-4-one;-   2-[(1H-Imidazol-1-yl) methyl]-3-phenyl-4H-chromen-4-one;-   2-[(9H-Purin-6-ylthio) methyl]-6-bromo-3-phenyl-4H-chromen-4-one;-   2-((4-Amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-6-bromo-3-phenyl-4H-Chromen-4-one;-   2-[(6-Amino-9H-purin-9-yl)    methyl]-6-bromo-3-(4-fluorophenyl)-4H-chromen-4-one;-   2-[(6-Amino-9H-purin-9-yl)    methyl]-3-(4-fluorophenyl)-4H-chromen-4-one;-   6-Bromo-3-(4-fluorophenyl)-2-(morpholinomethyl)-4H-chromen-4-one;-   6-Bromo-3-(4-fluorophenyl)-2-(morpholinomethyl)-4H-chromen-4-one    hydrochloride;-   3-(4-fluorophenyl)-2-(morpholinomethyl)-4H-chromen-4-one;-   3-(4-fluorophenyl)-2-(morpholinomethyl)-4H-chromen-4-one    hydrochloride;-   2-[(6-Amino-9H-purin-9-yl)methyl]-6-bromo-3-o-tolyl-4H-chromen-4-one;-   7-[(6-Bromo-4-oxo-3-phenyl-4H-chromen-2-yl)    methyl]-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione;-   2-(1-(6-Amino-9H-purin-9-yl)    ethyl)-6-bromo-3-phenyl-4H-chromen-4-one;-   2-(1-(9H-Purin-6-ylthio) ethyl)-6-bromo-3-phenyl-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl) ethyl)-3-phenyl-4H-chromen-4-one;-   (S)-2-(1-(9H-purin-6-ylamino)    ethyl)-6-bromo-3-phenyl-4H-chromen-4-one;-   2-((9H-purin-6-ylamino) methyl)-6-bromo-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-bromo-3-phenyl-4H-chromen-4-one;-   2-((6-Amino-9H-purin-9-yl)    methyl)-6-methoxy-3-phenyl-4H-chromen-4-one;-   2-(1-(6-Arnino-9H-purin-9-yl)    ethyl)-6-bromo-3-(2-fluorophenyl)-4H-chrornen-4-one;-   2-((6-Amino-9H-purin-9-yl)    methyl)-6-bromo-3-(2-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl)propyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((6-Amino-9H-purin-9-yl)    methyl)-3-(2-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl)    ethyl)-3-(2-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl)    propyl)-3-(2-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-amino-9H-purin-9-yl)    propyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl)    propyl)-3-(4-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-amino-9H-purin-9-yl)    propyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl)    ethyl)-3-(4-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl)    ethyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-Amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-phenyl-4H-chromen-4-one;-   2-((9H-purin-6-ylamino) methyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl) ethyl)-3-o-tolyl-4H-chromen-4-one;-   2-((9H-purin-6-ylamino) methyl)-3-(2-fluorophenyl)-4H-chromen-4-one;-   2-((9H-purin-6-ylamino) methyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(9H-purin-6-ylamino)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-amino-9H-purin-9-yl)    ethyl)-6-fluoro-3-(2-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-Amino-9H-purin-9-yl)    ethyl)-3-(3,5-difluorophenyl)-4H-chromen-4-one;-   2-(1-(6-amino-9H-purin-9-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-Amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   (R)-2-(1-(9H-purin-6-ylamino)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(9H-purin-6-ylamino)    ethyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   2-((4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    propyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(pyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-amino-3-(3-hydroxyprop-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-amino-3-(1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-amino-3-(3-(hydroxymethyl)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-   methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-amino-3-(1H-indazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-amino-3-(3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one-   2-((4-amino-3-(3-hydroxypropyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   N-(3-(4-amino-1-((4-oxo-3-phenyl-4H-chromen-2-yl)    methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl) acetamide;-   2-((4-amino-3-(3-fluoro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-amino-3-(3-fluoro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-hydroxy-3-methylbut-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(9H-purin-6-ylamino) ethyl)-3-phenyl-4H-chromen-4-one;-   (S)-2-(1-(9H-purin-6-ylamino)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1H-indazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3,5-dimethyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-(hydroxymethyl)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-fluoro-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-fluoro-3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-hydroxyprop-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-chloro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-chloro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-(trifluoromethoxy)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((6-amino-9H-purin-9-yl)    methyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-fluoro-2-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-((4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-aminopyrimidin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1H-indol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-chloro-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-chloro-3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-chloro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-chloro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3,4-dihydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-Amino-3-(3-methyl-1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   tert-butyl-(5-(4-amino-1-(1-(3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)    ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl) thiophen-2-yl)    methylcarbamate-   2-(1-(4-amino-3-(5-(aminomethyl)    thiophen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methyl-1H-indazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   N-(4-(4-amino-1-(1-(3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)    ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl) phenyl) acetamide;-   2-(1-(4-amino-3-(4-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2,3-dihydrobenzofuran-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-ethyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methyl-1H-indol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-methoxypyrimidin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   4-(4-amino-1-(1-(3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)    ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl) thiophene-2-carbaldehyde;-   2-(1-(4-amino-3-(5-(hydroxymethyl)    thiophen-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-methyl-1H-benzo[d]imidazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    propyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methyl-1H-indol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((6-amino-9H-purin-9-yl)    methyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   2-((6-amino-9H-purin-9-yl)    methyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3-fluoro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((9H-purin-6-ylamino)    methyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((9H-purin-6-ylamino) methyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   (R)-2-(1-(9H-purin-6-ylamino)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-6-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(3,5-difluoro-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3,5-difluoro-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (+)-2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   (−)-2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3,5-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-methoxy-3,5-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-fluoro-5-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1-benzyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-methylpyridin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(6-morpholinopyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(dibenzo[b,    d]furan-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(benzyloxy)-3-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-chloro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-(dimethylamino)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-ethoxy-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(trifluoromethoxy)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(3-(4-acetylphenyl)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(benzyloxy)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(dimethylamino)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(methylsulfonyl)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-ethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(benzo[b]thiophen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(5-chlorothiophen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3,5-dimethylisoxazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-propoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-ethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-chloro-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(6-fluoropyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(pyrimidin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-(methoxymethyl)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(6-hydroxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   6-Fluoro-3-(3-fluorophenyl)-2-(1-(4-methoxyphenylamino)    ethyl)-4H-chromen-4-one;-   2-(1-(4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-Chloro-1H-pyrazolo[3,4-b]pyridin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-Chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-Chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1,3-dimethyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2,3-dimethyl-2H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(6-methoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(benzo[b]thiophen-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2,4-dimethoxypyrimidin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   3-(4-amino-1-(1-(3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)    ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-cyclopropylbenzamide;-   2-(1-(4-amino-3-(3-(morpholine-4-carbonyl)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-(difluoromethoxy)    phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   5-(4-amino-1-(1-(3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)    ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl) furan-2-carbaldehyde;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(6-Amino-9H-purin-9-yl)    methyl)-3-(3-fluorophenyl)-5-methoxy-4H-chromen-4-one;-   2-((4-Amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-3-(3-fluorophenyl)-5-methoxy-4H-chromen-4-one;-   2-((4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    methyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3-fluoro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-((4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (+)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (−)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (+)-2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (−)-2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-amino-9H-purin-9-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-5-fluoro-3-(4-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-5-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(benzofuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(9H-purin-6-ylamino)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (+)-2-(1-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (−)-2-(1-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(difluoromethoxy)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-5-fluoro-3-(1H-pyrazol-4-yl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-(tetrahydro-2H-pyran-4-yloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-isopropyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-(piperidin-4-yloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-(2-hydroxyethylamino)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-(isopropylamino)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(dimethylamino)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-methyl-1H-benzo[d]imidazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-d]-pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(4-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(difluoromethoxy)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(4-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(difluoromethoxy)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-ethyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(benzo[b]thiophen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(dimethylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(piperidin-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(6-isopropoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one    4-methylbenzenesulfonate;-   2-(1-(4-amino-3-(3-methyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one    4-methylbenzenesulfonate;-   2-(1-(4-amino-3-(4-(1-benzhydrylazetidin-3-yloxy)-3-fluorophenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-(trifluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-(oxetan-3-yloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(pyrrolidin-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   N-(4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)isobutyramide;-   2-(1-(4-amino-3-(4-isobutylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-isopropoxy-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(5,6-dihydro-4H-1,3-oxazin-2-yl)phenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-methylbenzenesulfonamide;-   4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluoro-N-isopropylbenzamide;-   2-(1-(4-amino-3-(4-(5-(methylamino)-1,3,4-thiadiazol-2-yl)phenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   N-(4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzyl)methanesulfonamide;-   4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-isopropylbenzenesulfonamide;-   4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-cyclopropylbenzenesulfonamide;-   2-(1-(4-amino-3-(2-isopropoxypyrimidin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (R)/(S)-2-(1-(4-amino-3-(3-fluoro-4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzenesulfonamide;-   methyl    4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)thiophene-2-carboxylate;-   2-(1-(4-amino-3-(5-methylthiophen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   methyl    4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-3-fluorobenzoate;-   2-(1-(9H-purin-6-ylamino)propyl)-5-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-hydroxyprop-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)/(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one    4-methylbenzenesulfonate;-   (+)-2-(1-(9H-purin-6-ylamino)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one-   2-(1-(9H-purin-6-ylamino)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (R)/(S)-2-(1-(4-amino-3-(3-fluoro-4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-methoxy-3,5-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(methoxymethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(imidazo[1,2-a]pyridin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   tert-butyl    (5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)furan-2-yl)methylcarbamate;-   2-(1-(4-amino-3-(2,4-dimethylthiazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(5-(morpholinomethyl)thiophen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-(5-amino-1,3,4-thiadiazol-2-yl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (−)-2-(1-(9H-purin-6-ylamino)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(1,3-dimethyl-1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2,3-dimethyl-2H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   N-(4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)isobutyramide;-   N-(4-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)acetamide;-   2-(1-(4-(dimethylamino)-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   5-fluoro-2-(1-(3-(3-fluoro-4-isopropoxyphenyl)-4-(methylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   5-fluoro-2-(1-(3-(3-fluoro-4-isopropoxyphenyl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   N-(2-fluoro-4-(1-(1-(5-fluoro-3-(4-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)isobutyramide;-   N-(2-fluoro-4-(1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)isobutyramide;-   (S)/(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one    sulphate;-   (S)/(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)/(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one    camphorsulphonate;-   2-(1-(4-amino-3-(4-(difluoromethoxy)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(1H-pyrazol-4-yl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-phenyl-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(4-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(4-fluorophenyl)-4H-chromen-4-one;-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(4-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(4-(difluoromethoxy)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(4-fluorophenyl)-4H-chromen-4-one;-   (R)-2-(1-(4-amino-3-(4-(difluoromethoxy)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(4-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-(dimethylamino)-3-(3-fluoro-4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   5-fluoro-2-(1-(3-(3-fluoro-4-morpholinophenyl)-4-(methylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-phenyl-4H-chromen-4-one;-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-phenyl-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(4-(difluoromethoxy)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-phenyl-4H-chromen-4-one;-   (R)-2-(1-(4-amino-3-(4-(difluoromethoxy)-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-phenyl-4H-chromen-4-one;-   (+)-5-fluoro-2-(1-(3-(3-fluoro-4-isopropoxyphenyl)-4-(methylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   (−)-5-fluoro-2-(1-(3-(3-fluoro-4-isopropoxyphenyl)-4-(methylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-amino-2-fluoro-9H-purin-9-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-amino-2-fluoro-9H-purin-9-yl)ethyl)-5-fluoro-3-(4-fluorophenyl)-4H-chromen-4-one;-   5-fluoro-3-(4-fluorophenyl)-2-(1-(6-morpholino-9H-purin-9-yl)ethyl)-4H-chromen-4-one;-   5-fluoro-3-(4-fluorophenyl)-2-(1-(6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)ethyl)-4H-chromen-4-one;-   2-(1-(6-(dimethylamino)-9H-purin-9-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(6-(dimethylamino)-9H-purin-9-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   5-fluoro-3-(3-fluorophenyl)-2-(1-(3-(3-methyl-1H-indazol-6-yl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-chloro-4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (+)-2-(1-(4-amino-3-(4-isopropoxy-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (−)-2-(1-(4-amino-3-(4-isopropoxy-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)/(R)-5-fluoro-2-(1-(3-(3-fluoro-4-isopropoxyphenyl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-chloro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(2-methylbenzo[d]oxazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   5-fluoro-3-(3-fluorophenyl)-2-(1-(6-morpholino-9H-purin-9-yl)ethyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-5-morpholino-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-morpholino-3-phenyl-4H-chromen-4-one;-   6-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)isoindolin-1-one;-   5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)isoindolin-1-one;-   2-(1-(3-(4-acetyl-3-fluorophenyl)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   5-fluoro-3-(3-fluorophenyl)-2-(1-(6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)ethyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-chloro-4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (R)-2-(1-(4-amino-3-(3-chloro-4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   N-(3-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)methanesulfonamide;-   (S)-2-(1-(6-(dimethylamino)-9H-purin-9-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (R)-2-(1-(6-(dimethylamino)-9H-purin-9-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(9H-purin-6-ylamino)ethyl)-5-fluoro-3-(2-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(4-ethoxy-3-(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-5-fluoro-3-(3-fluorophenyl)-2-(1-(2-methoxy-9H-purin-6-ylamino)ethyl)-4H-chromen-4-one;-   (R)-5-fluoro-3-(3-fluorophenyl)-2-(1-(2-methoxy-9H-purin-6-ylamino)ethyl)-4H-chromen-4-one;-   (S)/(R)-5-fluoro-2-(1-(2-fluoro-9H-purin-6-ylamino)ethyl)-3-(3-fluoro    phenyl)-4H-chromen-4-one;-   (S)/(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-methyl-3-phenyl-4H-chromen-4-one;-   2-(1-(9H-purin-6-ylamino)ethyl)-5-fluoro-3-o-tolyl-4H-chromen-4-one;    and

pharmaceutically acceptable salts thereof.

In another preferred embodiment, the compound of formula (I) is a PI3KDelta inhibitor selected from

-   2-((6-amino-9H-purin-9-yl)methyl)-5-methyl-3-o-tolylquinazolin-4(3H)-one    (IC87114);-   (S)-2-(1-((9H-purin-6-yl)amino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one    (CAL-101, Idelalisib);-   INCB040093;-   AMG 319;-   (S)-2-(1-(9H-purin-6-ylamino)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one    (Compound C);-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and    pharmaceutically acceptable salts thereof.

In a further preferred embodiment, the compound of formula (I) is a dualPI3K Delta and Gamma inhibitor selected from

-   (S)-3-(1-((9H-purin-6-yl)    amino)ethyl)-8-chloro-2-phenylisoquinolin-1(2H)-one (IPI-145);-   (+)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one    (Compound A1);-   (−)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and pharmaceutically acceptable salts thereof.

In a preferred embodiment, the PDE-4 inhibitor is aprelimilast orroflumilast.

Yet another embodiment is a kit for treating an autoimmune, respiratoryor inflammatory disease or condition, the kit comprising:

(i) a PI3K Delta or PI3K Delta and Gamma inhibitor, and (ii) a PDE4inhibitor, either in a single pharmaceutical composition or in separatepharmaceutical compositions,

(ii) optionally, instructions for treating the autoimmune, respiratoryor inflammatory disease or condition with the PI3K Delta or PI3K Deltaand Gamma inhibitor, and PDE4 inhibitor, and

(iii) optionally, a container for placing the pharmaceutical compositionor pharmaceutical compositions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the effect of Compound A and its combination withCompound B at 30 and 300 nM on inhibition of TNFα release from U-937cells. **p<0.01 and ***p<0.001 (versus Compound A).

FIG. 2 depicts the effect of Compound A and its combination withCompound B at 300 nM on MMP-9 release from THP-1 cells.

FIG. 3 depicts the effect of Compound A with and without Compound B (30nM) on pAkt (S473) expression in differentiated U937 cells stimulatedwith Cigarette Smoke Extract (CSE).

FIG. 4 depicts the effect of Compound A and its combination withCompound B (300 nM) on the inhibition of release of elastase from humanneutrophils. **p<0.01 and ***p<0.001 (versus Compound A).

FIG. 5 depicts the effect of Compound A and its combination withCompound B (300 nM) on the proliferation of A549 cells after CSEinduction. * represents p<0.05, ** represents p<0.01 and *** representsp<0.001 when compared to administration of Compound A alone.

FIG. 6 depicts the inhibition of apoptosis in A549 cells by Compound Aand its combination with Compound B (300 nM).

FIGS. 7A, 7B, and 7C depict the inhibition in neutrophil infiltration byCompound A, Compound C and their combination, respectively.

FIGS. 8A, 8B, and 8C depict the inhibition in neutrophil infiltration byCompound A, Compound A1 and their combination.

FIG. 9 depicts the inhibition in macrophage infiltration for Compound A,Compound C and their combination in acute cigarette smoke induced cellinfiltration in Male Balb/c mice.

FIG. 10 depicts the inhibition in macrophage infiltration for CompoundA, Compound A1 and their combination in acute cigarette smoke inducedcell infiltration in Male Balb/c mice.

FIG. 11 depicts the inhibition in macrophage infiltration for CompoundA, Compound C and their combination in chronic cigarette smoke inducedcell infiltration in Male Balb/c mice.

DETAILED DESCRIPTION OF THE INVENTION Definitions

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange may vary from, for example, between 1% and 15% of the statednumber or numerical range. The term “comprising” (and related terms suchas “comprise” or “comprises” or “having” or “including”) includes thoseembodiments, for example, an embodiment of any composition of matter,composition, method, or process, or the like, that “consist of” or“consist essentially of” the described features.

The following abbreviations and terms have the indicated meaningsthroughout: PI3-K=Phosphoinositide 3-kinase; PI=phosphatidylinositol;and MeI=Methyl Iodide.

Abbreviations used herein have their conventional meaning within thechemical and biological arts, unless otherwise indicated.

The terms “substituted or unsubstituted”, “alkyl”, “alkoxy”, “alkenyl”,“alkynyl”, “aryl”, “arylalkyl”, “cycloalkyl”, “cycloalkylalkyl”,“cycloalkenylalkyl”, “cycloalkenyl”, “heteroaryl”, “heteroarylalkyl”,“heterocyclic ring” (or heterocyclyl), and “heterocyclylalkyl” are asdefined in International Patent Application Nos. PCT/IB2010/002804 andPCT/US2012/36594. Suitable pharmaceutically acceptable salts of the PI3Kinhibitors described herein include those described in InternationalPatent Application Nos. PCT/IB2010/002804 and PCT/US2012/36594.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment, as defined below. Thetherapeutically effective amount may vary depending upon the intendedapplication (in vitro or in vivo), or the subject and disease conditionbeing treated, e.g., the weight and age of the subject, the severity ofthe disease condition, the manner of administration and the like, whichcan readily be determined by one of ordinary skill in the art. The termalso applies to a dose that will induce a particular response in targetcells, e.g., reduction of platelet adhesion and/or cell migration. Thespecific dose will vary depending on the particular compounds chosen,the dosing regimen to be followed, whether it is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichit is carried.

As used herein, the terms “treatment” and “treating” refer to anapproach for obtaining beneficial or desired results including, but notlimited to, therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

A “therapeutic effect,” as that term is used herein encompasses atherapeutic benefit and/or a prophylactic benefit as described above. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

The term “subject” or “patient” refers to an animal, such as a mammal,for example a human. The methods described herein can be useful in bothhuman therapeutics and veterinary applications. In some embodiments, thepatient is a mammal, and in some embodiments, the patient is human. Forveterinary purposes, the term “subject” and “patient” include, but arenot limited to, farm animals including cows, sheep, pigs, horses, andgoats; companion animals such as dogs and cats; exotic and/or zooanimals; laboratory animals including mice, rats, rabbits, guinea pigs,and hamsters; and poultry such as chickens, turkeys, ducks, and geese.

The term “selective inhibition” or “selectively inhibit” as applied to abiologically active agent refers to the agent's ability to selectivelyreduce the target signaling activity as compared to off-target signalingactivity, via direct or indirect interaction with the target.

As used herein, the term “PI3-kinase δ selective inhibitor” generallyrefers to a compound that inhibits the activity of the PI3-kinase δisozyme more effectively than other isozymes of the PI3K family (alpha,beta, and gamma). For instance, the PI3-kinase δ selective inhibitor mayrefer to a compound that exhibits a 50% inhibitory concentration (IC50)with respect to the delta type I PI3-kinase that is at least 10-fold, atleast 20-fold, at least 50-fold, at least 100-fold, or lower, than theinhibitor's IC50 with respect to the rest of the other type IPI3-kinases (i.e., alpha, beta, and gamma).

As used herein, the term “Dual PI3-kinase Delta (δ) and Gamma (γ)inhibitor” generally refers to a compound that inhibits the activity ofboth the PI3-kinase δ and y isozymes more effectively than otherisozymes of the PI3K family A PI3-kinase δ and y dual inhibitor compoundis therefore more selective for PI3-kinase δ and y than conventionalPI3K inhibitors such as wortmannin and LY294002, which are“non-selective PI3K inhibitors.”

For instance, the Dual PI3-kinase δ and y selective inhibitor may referto a compound that exhibits a 50% inhibitory concentration (IC50) withrespect to the delta and gamma type I PI3-kinase that is at least10-fold, at least 20-fold, at least 50-fold, at least 100-fold, orlower, than the inhibitor's IC50 with respect to the rest of the othertype I PI3-kinases (i.e., alpha, and beta).

The therapeutic methods of the invention include methods for thetreatment of conditions associated with an inflammatory response. An“Inflammatory response” is characterized by redness, heat, swelling andpain (i.e., inflammation) and typically involves tissue injury ordestruction. An inflammatory response is usually a localized, protectiveresponse elicited by injury or destruction of tissues, which serves todestroy, dilute or wall off (sequester) both the injurious agent and theinjured tissue. Inflammatory responses are notably associated with theinflux of leukocytes and/or leukocyte (e.g., neutrophil) chemotaxis.Inflammatory responses may result from infection with pathogenicorganisms and viruses, noninfectious means such as trauma or reperfusionfollowing myocardial infarction or stroke, immune responses to foreignantigens, and autoimmune diseases. Inflammatory responses amenable totreatment with the methods and compounds according to the inventionencompass conditions associated with reactions of the specific defensesystem as well as conditions associated with reactions of thenon-specific defense system.

The therapeutic methods of the invention include methods for thetreatment of conditions associated with inflammatory cell activation.“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatibility antigens or cell adhesion molecules) ininflammatory cells (including, but not limited to, monocytes,macrophages, T lymphocytes, B lymphocytes, granulocytes(polymorphonuclear leukocytes including neutrophils, basophils, andeosinophils) mast cells, dendritic cells, Langerhans cells, andendothelial cells). It will be appreciated by persons skilled in the artthat the activation of one or a combination of these phenotypes in thesecells can contribute to the initiation, perpetuation, or exacerbation ofan inflammatory condition.

“Autoimmune disease” as used herein refers to any group of disorders inwhich tissue injury is associated with humoral or cell-mediatedresponses to the body's own constituents.

An “allergic” disease generally refers to any symptoms, tissue damage,or loss of tissue function resulting from allergy

An “arthritic” disease generally refers to any disease that ischaracterized by inflammatory lesions of the joints attributable to avariety of etiologies.

“Dermatitis” generally refers to any of a large family of diseases ofthe skin that are characterized by inflammation of the skin attributableto a variety of etiologies.

The term “co-administration,” “administered in combination with,” andtheir grammatical equivalents, as used herein, encompassesadministration of two or more agents to an animal so that both agentsand/or their metabolites are present in the animal at the same time.Co-administration includes simultaneous administration in separatecompositions, administration at different times in separatecompositions, or administration in a composition in which both agentsare present.

The term “pharmaceutically acceptable salts” as used herein includessalts derived from inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu,Zn, and Mn; salts of organic bases such as N,N′-diacetylethylenediamine,glucamine, triethylamine, choline, hydroxide, dicyclohexylamine,metformin, benzylamine, trialkylamine, and thiamine; salts of chiralbases such as alkylphenylamine, glycinol, and phenyl glycinol; salts ofnatural amino acids such as glycine, alanine, valine, leucine,isoleucine, norleucine, tyrosine, cystine, cysteine, methionine,proline, hydroxy proline, histidine, omithine, lysine, arginine, andserine; quaternary ammonium salts of the compounds of invention withalkyl halides, alkyl sulphates such as MeI and (Me)₂SO₄; salts ofnon-natural amino acids such as D-isomers or substituted amino acids;salts of guanidine; and salts of substituted guanidine wherein thesubstituents are selected from nitro, amino, alkyl, alkenyl, alkynyl,ammonium or substituted ammonium salts and aluminum salts. Salts mayinclude acid addition salts where appropriate which are sulphates,nitrates, phosphates, perchlorates, borates, hydrohalides, acetates,tartrates, maleates, citrates, fumarates, succinates, palmoates, methanesulphonates, benzoates, salicylates, benzenesulfonates, ascorbates,glycerophosphates, and ketoglutarates.

PI3K Delta and dual PI3K Delta and Gamma Inhibitors

Examples of PI3-kinase δ selective inhibitors and dual PI3-kinase delta(δ) and gamma (γ) inhibitors that may be used in the compositions andmethods described herein include, but are not limited to, CAL-101(idelalisib), IPI-145 (duvelisib) and the compounds disclosed inInternational Publication No. WO 2012/151525, U.S. Patent PublicationNos. 2011/0118257 and 2012/0289496, International Patent ApplicationNos. PCT/IB2010/002804, filed Nov. 3, 2010, PCT/US2012/36594, filed May4, 2012, PCT/US2013/055434, filed Jul. 2, 2013 and U.S. patentapplication Ser. No. 13/933,856, filed Jul. 2, 2013. Additional nonlimiting examples are also disclosed in International Publication Nos.WO 2001/081346, WO 2003/035075, WO 2005/113554, WO 200/113556, WO2006/024666, WO 2008/118454, WO 2008/118455, WO 2009/010530, WO2009/064802, WO 2009/088986, WO 2009/088990, WO 2009/147189, WO2010/005558, WO 2010/051042, WO 2010/051043, WO 2010/056320, WO2010/057048, WO 2010/092015, WO 2010/092962, WO 2010/096389, WO2010/102958, WO 2010/110685, WO 2010/110686, WO 2010/111432, WO2010/123931, WO 2010/135014, WO 2010/136491, WO 2010/138589, WO2010/144513, WO 2010/151737, WO 2010/151740, WO 2010/151791, WO2011/005119, WO 2011/008302, WO 2011/008487, WO 2011/011550, WO2011/012883, WO 2011/021038, WO 2011/022439, WO 2011/041399, WO2011/041634, WO 2011/048111, WO 2011/048936, WO 2011/055215, WO2011/075268, WO 2011/075630, WO 2011/075643, WO 2011/101429, WO2011/123751, WO 2011/130342, WO 2011/156759, WO 2011/163195, WO2012/003262, WO 2012/003264, WO 2012/003271, WO 2012/003274, WO2012/003278, WO 2012/003283, WO 2012/004299, WO 2012/007493, WO2012/0135009, WO 2012/020762, WO 2012/021696, WO 2012/032067, WO2012/037204, WO 2012/037226, WO 2012/040634, WO 2012/044641, WO2012/052753, WO 2012/055846, WO 2012/061696, WO 2012/064973, WO2012/068343, WO 2012/087784, WO 2012/087881, WO 2012/097000, WO2012/107465, WO 2012/116237, WO 2012/121953, WO 2012/125510, WO2012/125629, WO 2012/126901, WO 2012/135160, WO 2012/135166, WO2012/135175, WO 2012/140419, WO 2012/146666, WO 2012/146667, WO2012/148548, WO 2012/151525, US 2012/0220575, US 2012/0238587, WO2013/012915, WO 2013/012918, WO 2013/032591, WO 2013/033569, WO2013/052699, WO 2013/057711, WO 2013/067141, WO 2013/067306, WO2013/071264, WO 2013/078441, WO 2013/082540, WO 2013/090725, WO2013/116562, WO 2013/132270, WO 2013/134288, WO 2013/136075 and WO2013/136076. Each of these publications describing PI3K inhibitors andtheir preparation are incorporated herein by reference.

PDE-4 Inhibitors

Suitable PDE-4 inhibitors for use in the compositions and methodsdescribed herein include, but are not limited to, enprofylline,theophylline, aminophylline, oxtriphylline, apremilast, roflumilast,ariflo (cilomilast), tofimilast, pumafentrine, lirimilast, arofylline,atizorame, oglemilastum, D-4418, Bay-198004, BY343, CP-325,366, D-4396(Sch-351591), AWD-12-281 (GW-842470), NCS-613, CDP-840, D-4418,PD-168787, T-440, T-2585, V 1 1294A, CI-1018, CDC-801, CDC-3052,D-22888, YM-58997, Z-15370,N-(3,5-dichloro-1-oxo-pyridin-4-yl)-4-difluoromethoxy-3-cyclopropylmethoxybenzamide,(−)-p-[(4 aR*, 10bS*)-9-ethoxy-1,2,3,4,4a,10b-hexahydro-8-methoxy-2-methylbenzo[s][1,6]-naphthyridin-6-yl]-N,N-diiso-propylbenzamid(R)-(+)-1-(4-bromobenzyl)-4-[(3-cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidon,3-(cyclopentyloxy-4-methoxyphenyl)-1-(4-N′-[N-2-cyano-S-methyl-isothioureido]benzyl)-2-pyrrolidone, cis[4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl) cyclohexan-1-carboxylic acid],2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one,cis[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxy phenyl)cyclohexan-1-ol], (R)-(+)-ethyl[4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidin-2-yliden]acetate,(S)-(−)-ethyl[4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidin-2-yliden]acetate,9cyclopentyl-5,6-dihydro-7-ethyl-3-(2-thienyl)-9H-pyrazolo[3,4c]-1,2,4-triazolo[4,3a]pyridine and9-cyclopentyl-5,6-dihydro-7-ethyl-3-(tert-butyl)-9H-pyrazolo[3,4c]-1,2,4-triazolo[4,3 a]pyridine, optionally in racemic form, asenantiomers, diastereomers or as pharmacologically acceptable salts,solvates or hydrates or the compounds as disclosed in the WO 2012/016845and WO 2012/016889.

In a preferred embodiment, the PDE4 inhibitor is selected fromtheophylline, aminophylline, oxtriphylline, roflumilast and apremilast.In a further preferred embodiment, the PDE4 inhibitor is roflumilast. Inyet another embodiment, the PDE4 inhibitor is apremilast.

Pharmaceutical Compositions

In one aspect, the present invention provides a pharmaceuticalcomposition comprising a PI3K Delta or dual PI3K Delta and Gamma dualinhibitor and a PDE4 inhibitor, and, optionally, one or morepharmaceutically acceptable carriers or excipients.

In one embodiment, the pharmaceutical composition includes atherapeutically effective amount of PI3K Delta or dual PI3K Delta andGamma inhibitor and a therapeutically effective amount of PDE4inhibitor. In another embodiment, the pharmaceutical compositionincludes a synergistic effective amount of (i) PI3K Delta or dual PI3KDelta and Gamma inhibitor and (ii) PDE4 inhibitor. For example, thepharmaceutical composition may include from about 0.1 μg to about 2 gm,preferably from about 1 μg to about 1000 mg, more preferably from about10 μg to about 500 mg, such as from about 100 μg to about 100 mg, of thePI3K Delta or dual PI3K Delta and Gamma inhibitor, and from about 0.1 μgto about 500 mg, preferably from about 1 μg to about 100 mg, morepreferably from about 10 μg to about 50 mg, such as from about 10 μg toabout 10 mg of the PDE4 inhibitor.

The pharmaceutical composition may further include one or moreadditional active ingredients, such as those useful for the preventionand/or treatment of a respiratory disease, such as beta2-agonists (e.g.,salbutamol, salmeterol, and vilanterol); corticosteroids (such asfluticasone propionate or furoate, flunisolide, mometasone furoate,rofleponide and ciclesonide); and anticholinergic or antimuscarinicagents (such as ipratropium bromide, oxytropium bromide, tiotropiumbromide, and oxybutynin); and combinations thereof.

The pharmaceutical carriers and/or excipients may be selected fromdiluents, fillers, salts, disintegrants, binders, lubricants, glidants,wetting agents, controlled release matrices, colorants, flavourings,buffers, stabilizers, solubilizers, and combinations thereof.

The pharmaceutical compositions of the present invention can beadministered alone or in combination with one or more additional activeingredients, such as those described above. The pharmaceuticalcompositions of the present invention can be administered together or ina sequential manner with one or more of the other active ingredients.Where desired, the pharmaceutical composition of the present inventionand other active ingredients may be co-administered or both may beadministered in a sequence to use them as a combination.

The compounds and pharmaceutical compositions of the present inventioncan be administered by any route that enables delivery of the compoundsto the site of action, such as, but not limited to, orally,intranasally, topically (e.g., transdermally), intraduodenally,parenterally (including intravenously, intraarterially,intramuscularally, intravascularally, intraperitoneally or by injectionor infusion), intradermally, by intramammary, intrathecally,intraocularly, retrobulbarly, intrapulmonary (e.g., aerosolized drugs)or subcutaneously (including depot administration for long term releasee.g., embedded-under the-splenic capsule, brain, or in the cornea),sublingually, anally, rectally, vaginally, or by surgical implantation(e.g., embedded under the splenic capsule, brain, or in the cornea) orby inhalation.

The compositions can be administered in solid, semi-solid, liquid orgaseous form, or may be in dried powder, such as lyophilized form. Thepharmaceutical compositions can be packaged in forms convenient fordelivery, including, for example, solid dosage forms such as capsules,sachets, cachets, gelatins, papers, tablets, suppositories, pellets,pills, troches, and lozenges. The type of packaging will generallydepend on the desired route of administration. Implantable sustainedrelease formulations are also contemplated, as are transdermalformulations. In one preferred embodiment, the pharmaceuticalcomposition is a solid oral dosage form, such as a tablet or capsule.

In another embodiment, the pharmaceutical composition is suitable forinhalation (e.g., by being aerosolized).

The present invention further relates to a pharmaceutical compositionaccording to any embodiment described herein for use in the treatment ofautoimmune, respiratory and/or inflammatory diseases and conditions.

Another embodiment of the present invention relates to a method oftreating autoimmune respiratory and/or inflammatory diseases andconditions, comprising administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositionaccording to any of the embodiments described herein.

Another embodiment of the present invention relates to the use of apharmaceutical composition according to any of the embodiments describedherein for the manufacture of a medicament for treating autoimmune,respiratory and/or inflammatory diseases and conditions in a subject inneed thereof.

In the pharmaceutical compositions according to any of the embodimentsdescribed herein, the PI3K Delta inhibitor may be in the form of asolvate, a hydrate or as salt with pharmacologically acceptable acid orbase.

In the pharmaceutical compositions according to any of the embodimentsdescribed herein, the Dual PI3K Delta and Gamma inhibitor may be in theform of a solvate, a hydrate or as salt with pharmacologicallyacceptable acid or base.

In the pharmaceutical compositions according to any of the embodimentsdescribed herein, the PDE4 inhibitor may be may be in the form of asolvate, a hydrate or as salt with pharmacologically acceptable acid orbase.

In one embodiment, the present invention relates to a pharmaceuticalcomposition according to any of the embodiments described herein,wherein the PDE4 inhibitor is roflumilast.

Another particular embodiment of the present invention relates topharmaceutical compositions according to any of the embodimentsdescribed herein, wherein the PDE4 inhibitor is aprelimilast.

Yet another particular embodiment of the present invention relates topharmaceutical compositions according to any of the embodimentsdescribed herein, wherein the PDE4 inhibitor is theophylline.

Methods of Treatment

Another embodiment of the present invention is a method of treating animmune system-related disease (e.g., an autoimmune disease), a diseaseor disorder involving inflammation (e.g., asthma, chronic obstructivepulmonary disease, rheumatoid arthritis, inflammatory bowel disease,glomerulonephritis, neuroinflammatory diseases, multiple sclerosis,uveitis and disorders of the immune system), cancer or otherproliferative disease, a hepatic disease or disorder, or a renal diseaseor disorder. The method includes administering an effective amount of aPI3K Delta or dual PI3K Delta and Gamma dual inhibitor and a PDE4inhibitor.

The PI3K Delta or dual PI3K Delta and Gamma dual inhibitor and PDE4inhibitor (and optionally other active ingredients) can be incorporatedin a single pharmaceutical composition and administered, oralternatively, can be administered in separate pharmaceuticalcompositions, which can be administered at the same time or at differenttimes.

The amount of each compound to be administered depends on the subject(such as a mammal or human in particular) being treated, the severity ofthe disorder or condition, the rate of administration, the dispositionof the compound and the discretion of the prescribing physician. In oneembodiment, the effective dosage for the PI3K Delta or PI3K Delta andGamma dual inhibitor is from about 0.001 to about 100 mg per kg bodyweight per day, for example, from about 1 to about 35 mg/kg/day, insingle or divided doses. For a 70 kg human, this would amount to about0.05 to 7 g/day, preferably about 0.05 to about 2.5 g/day. In oneembodiment, the effective dosage for the PDE4 inhibitor is from about0.001 to about 100 mg per kg body weight per day, for example, fromabout 0.1 to about 100 mcg/kg/day, in single or divided doses (e.g.,from about 7 to about 7000 mcg/day or from about 10 to about 1000mcg/day). An effective amount of the PI3K Delta or PI3K Delta and Gammadual inhibitor and/or PDE4 inhibitor may be administered in eithersingle or multiple doses (e.g., twice or three times a day).

In another embodiment, the PI3K Delta or dual PI3K Delta and Gammainhibitor and at the PDE4 inhibitor are each administered in an amountranging from about 0.01 mg to about 1000 mg.

In yet another embodiment, from about 0.05 mg to about 7 g of the PI3KDelta or PI3K Delta and Gamma dual inhibitor and from about 7 to about7000 mcg (e.g., from about 10 to about 1000 mcg) of the PDE4 inhibitorare administered daily. For example, when the PDE4 inhibitor isroflumilast, from about 100 to about 2000 mcg of roflumilast isadministered daily (preferably orally or by inhalation). In anotherembodiment, about 200, 300, 400, 500, or 600 mcg of roflumilast isadministered daily (preferably orally or by inhalation).

The PI3K Delta or PI3K Delta and Gamma dual inhibitor and PDE4 inhibitormay be administered orally or by inhalation. In one embodiment, the PI3KDelta or PI3K Delta and Gamma dual inhibitor is administered byinhalation and the PDE4 inhibitor is administered orally. In anotherembodiment, the PI3K Delta or PI3K Delta and Gamma dual inhibitor andthe PDE4 inhibitor are both administered orally. In yet anotherembodiment, the PI3K Delta or PI3K Delta and Gamma dual inhibitor isadministered orally and the PDE4 inhibitor is administered byinhalation. In another embodiment, the PI3K Delta or PI3K Delta andGamma dual inhibitor and the PDE4 inhibitor are both administered byinhalation.

In additional embodiments of any of the methods described herein, thePI3K Delta or dual PI3K Delta and Gamma inhibitor and at the PDE4inhibitor are administered either as oral or by inhalation For example,Both the PI3K Delta or dual PI3K Delta and Gamma inhibitor can and thePDE4 inhibitor is administered orally. Both the PI3K Delta or dual PI3KDelta and Gamma inhibitor can and the PDE4 inhibitor is administered byinhalation, One of the PI3K Delta or dual PI3K Delta and Gamma inhibitoris administered orally and the PDE4 inhibitor is administered byinhalation or One of the PI3K Delta or dual PI3K Delta and Gammainhibitor is administered by inhalation and the PDE4 inhibitor isadministered orally.

In additional embodiments of any of the methods described herein, thePI3K Delta or dual PI3K Delta and Gamma inhibitor and the PDE4 inhibitorare administered at a ratio of about 1:100 to about 100:1 by weight.

In one embodiment, the therapeutically effective amount of PI3K Delta ordual PI3K Delta and Gamma inhibitor is administered twice daily to onceevery three weeks, and the therapeutically effective amount of the PDE4inhibitor is administered twice daily to once every three weeks.

Examples of immune disorders which can be treated by the compounds ofthe present invention include, but are not limited to, psoriasis,rheumatoid arthritis, vasculitis, inflammatory bowel disease,dermatitis, osteoarthritis, asthma, inflammatory muscle disease,allergic rhinitis, vaginitis, interstitial cystitis, scleroderma,osteoporosis, eczema, allogeneic or xenogeneic transplantation (organ,bone marrow, stem cells and other cells and tissues) graft rejection,graft-versus-host disease, lupus erythematosus, inflammatory disease,type I diabetes, pulmonary fibrosis, dermatomyositis, Sjogren'ssyndrome, thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis),myasthenia gravis, autoimmune hemolytic anemia, multiple sclerosis,cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis,allergic conjunctivitis and atopic dermatitis.

A further embodiment of the present invention relates to a method oftreating an disease or disorder selected from respiratory diseases andconditions such as diseases of the airways and lungs which areaccompanied by increased or altered production of mucus and/orinflammatory and/or obstructive diseases of the airways such as acutebronchitis, chronic bronchitis, chronic obstructive bronchitis (COPD),cough, pulmonary emphysema, allergic or non-allergic rhinitis orsinusitis, chronic sinusitis or rhinitis, nasal polyposis, chronicrhinosinusitis, acute rhinosinusitis, asthma, allergic bronchitis,alveolitis, Farmer's disease, hyperreactive airways, bronchitis orpneumonitis caused by infection, e.g. by bacteria or viruses orhelminthes or fungi or protozoons or other pathogens, pediatric asthma,bronchiectasis, pulmonary fibrosis, adult respiratory distress syndrome,bronchial and pulmonary edema, bronchitis or pneumonitis or interstitialpneumonitis caused by different origins, e.g. aspiration, inhalation oftoxic gases, vapors, bronchitis or pneumonitis or interstitialpneumonitis caused by heart failure, X-rays, radiation, chemotherapy,bronchitis or pneumonitis or interstitial pneumonitis associated withcollagenosis, e.g. lupus erythematodes, systemic scleroderma, lungfibrosis, idiopathic pulmonary lung fibrosis (IPF), interstitial lungdiseases or interstitial pneumonitis of different origin, includingasbestosis, silicosis, M. Boeck or sarcoidosis, granulomatosis, cysticfibrosis or mucoviscidosis, or a-1-antitrypsin deficiency; or selectedfrom inflammatory diseases and conditions such as inflammatory diseasesof the gastrointestinal tract of various origins such as inflammatorypseudopolyps, Crohn's disease, ulcerative colitis, inflammatory diseasesof the joints, such as rheumatoid arthritis, or allergic inflammatorydiseases of the oro-nasopharynx, skin or the eyes, such as atopicdermatitis, seasonal and perenial, chronic uritcaria, hives of unknowncause and allergic conjunctivitis; and in particular selected fromasthma, allergic and non-allergic rhinitis, COPD and atopic dermatitis;comprising administering a therapeutically effective amount of apharmaceutical composition according to any of the embodiments describedherein to a patient in need thereof.

A further embodiment of the present invention relates to the use of apharmaceutical composition according to any of the embodiments describedherein for the manufacture of a medicament for treating respiratoryand/or inflammatory diseases and conditions, particularly wherein therespiratory and/or inflammatory diseases or conditions are selected fromasthma, allergic and non-allergic rhinitis, COPD and atopic dermatitis.

A further embodiment of the present invention relates to apharmaceutical composition according to any of the embodiments describedherein for use in the treatment of respiratory and inflammatory diseasesand conditions, particularly wherein the respiratory and inflammatorydiseases or conditions are selected from asthma, allergic andnon-allergic rhinitis, COPD and atopic dermatitis.

The present invention is now further illustrated by means of thefollowing non-limiting examples.

EXAMPLES

As described in the following examples, Compound A is roflumilast;Compound B is IC87114; Compound C is Example 74 of InternationalPublication No. WO 11/055215 (PCT/2010/002804) and Compound A1 isExample 7 of International Publication No. WO 2012/151525 (PCT/US2012/036594). The representative examples use roflumilast andaprelimilast as the PDE-4 inhibitor.

Example 1 Combination Studies of a PI3K Delta Inhibitor and a PDE-4Inhibitor

Compound A was used as the PDE4 inhibitor and compounds B and C wereused as the PI3K Delta inhibitor in these studies.

Estimation of TNFα:

U937 cells were plated at 100,000 cells per well in a 96 well plate andincubated with the desired concentration of compounds for 30 minutesprior to the addition of 1 μg/ml LPS. Supernatants were collected after24 hours and estimated for TNFα concentration by ELISA as per theprotocol recommended by the kit manufacturer (eBioscience, USA).Briefly, Nunc Maxisorp plates were coated with 100 μl of 1 mg/ml TNFαcoating antibody. Supernatant was transferred to plates and incubated at37° C. for 2 h. Anti-TNFα detection antibody and avidin-HRP were addedfollowed by TMB substrate. Absorbance was measured at 450 nm on FluostarOmega (BMG Labtech, NC, USA).

Results:

The results are shown in FIG. 1. Combining 10 μM of Compound A(roflumilast) with 300 or 30 nM of Compound B (IC87114) effectivelyreduced TNFα release with an Emax of 75% and 58% respectively.Significant reductions (p<0.01) in TNFα secretion were observed withconcentrations as low as 300 nM of Compound A combined with 300 nMCompound B indicating that roflumilast in the presence of a PI3Kδinhibitor controls the regulation of cytokines responsible forexacerbation of COPD.

Quantification of Matrix Metalloproteinase (MMP)-9:

Gelatinase activity of MMP-9 was determined by zymography. Proteins insupernatant (after incubation of THP-1 cells with 50 ng PMA for 24 h)were separated on gels containing 0.1% gelatin (Sigma, USA). Gels werere-natured by incubation in 2.5% Triton X-100 for 30 mM, incubatedovernight in substrate buffer (50 mMTris-HCl, pH 7.5) containing 10 mMCaCl₂) and 0.05% ZnCl₂ at 37° C. and stained with Coomassie brilliantblue (0.5%). Clear areas in the blue background of the gels demonstratedthe presence of gelatinase activity. Molecular weight markers(Fermentas, Lithuania) were run with each gel. Band intensity wascalculated using ImageJ 1.42 (NIH, USA).

Results:

The results are shown in FIG. 2. A two fold increase in MMP-9 releasedue to induction was observed compared to the blank wells. The IC50 ofCompound A for the reduction of MMP-9 was 577 nM while Compound B alonehad no effect on the MMP-9 release at 300 nM. However, the presence ofCompound B (300 nM) with Compound A, effectively potentiated the effectof Compound A even at the lowest concentration tested (0.1 nM).

SDS-PAGE for the Estimation of Phospho-Akt:

U-937 monocytes were differentiated into macrophages by incubation with50 ng/ml PMA for 48 h. Cells were trypsinized and plated at a density of100,000 per well and stimulated with CSE for 2 h in starvation media.Lysates were made using RIPA buffer and run by SDS-PAGE, transferred toPVDF membrane and probed with phospho-Akt (S473) antibody (Cellsignaling, USA) followed by anti-rabbit IgG (Cell signaling, USA). Bandintensity was calculated using ImageJ 1.42 (NIH, USA).

Results:

The results are shown in FIG. 3. Individually, Compound A and Compound Bdose-dependently inhibited Akt phosphorylation with IC₅₀ values of 1934and 780 nM, respectively. However, in combination with 30 nM of CompoundB, a significant reduction (˜1290 fold) in the IC₅₀ of Compound A (1.5nM) was observed, indicating accentuation of the response in line withMMP-9 reduction.

Quantification of Neutrophil Elastase:

Blood was procured from a local blood bank. Neutrophils were obtainedusing the dextran sedimentation method. Cells were plated at 100,000cells per well in a 96-well plate, treated with fMLP (1 μM) andN-Succinyl-Ala-Ala-Ala-p-nitroanalide (1 μM) (Sigma) and incubated for 2h. Intensity of digested substrate after incubation was determined bymeasuring the absorbance at 405 nm on a Fluostar Omega (BMG Labtech, NC,USA).

Results:

The results are shown in FIG. 4. Compound A inhibited fMLP inducedelastase release with an IC₅₀ of 10.2 nM (Emax=43%) while the reductionwith Compound B alone was negligible (10% at 300 nM). Combining CompoundA with 300 nM of Compound B resulted in an increase in Emax (65%) alongwith a corresponding reduction in IC₅₀ of Compound A to 4.8 nM.

Cell Proliferation Assay:

A549 cells were plated at 10,000 cells per well in a 96-well plate andincubated with CSE for 72 h. Viability of cells was determined byestimating the amount of soluble formazan (in DMSO) formed afteraddition of 100 μg MTT and a 4 h incubation at 37° C. Media was removedand the crystals were dissolved in 100 μl DMSO. Absorbance was measuredat 450 nm on a Fluostar Omega (BMG Labtech, NC, USA).

Results:

The results are shown in FIG. 5. The combination of Compound A and 300nM of Compound B caused a marked reversal of the anti-proliferativeeffect of CSE with an IC₅₀ of 8.7 nM. Without wishing to be bound by anyparticular theory, the inventors theorize that the combination of a PDE4inhibitor and a PI3Kδ inhibitor serves to protect the lung alveolarepithelium, thereby minimizing the progression of COPD.

Cell Cycle Analysis:

A549 cells were plated in 6-well plates at a density of 100,000 cellsper well and stimulated with CSE for 72 h. After incubation, cells werefixed in 70% ethanol and stored at 4° C. till analysis. Cells werestained with Guava Cell Cycle reagent according to the manufacturer'sinstructions. Cell cycle data were obtained using the Guava PersonalCell Analysis System (Millipore, USA).

Results:

The results are shown in FIG. 6. Treatment with Compound A resulted in adose-dependent inhibition of apoptosis. This decrease was significantlyfurther potentiated using a combination of Compound A with 300 nM ofCompound B, indicating the potential utility of this combination inpreventing lung damage.

In summary, the inhibition of TNFα, pAkt, and MMP-9 in differentiatedU937 macrophages upon stimulation with LPS/CSE was determined.Neutrophil functionality manifested by a modulation of elastase activitywas estimated. The protective effect of the combination (Compounds A andB) on CSE induced apoptosis of lung epithelial cells was alsodetermined. The data demonstrates that the combination of Compound A(PDE4 inhibitor) and Compound B (PI3K delta inhibitor) reduces TNFα,pAkt and MMP-9 at nanomolar concentrations and is several fold morepotent than either of the compounds alone. Inhibition of neutrophilelastase was also increased significantly with the combination, therebyproviding evidence for the therapeutic benefits of the combination of aPI3K delta inhibitor and a PDE4 inhibitor in the treatment of COPD.

LPS Induced TNFα in Human Whole Blood (HWB):

Freshly collected HWB was diluted with media and incubated with thedesired concentration of inhibitor for 15 min. LPS (1 μg/ml) was addedand then incubated for 24 hours. The supernatant was collected and TNFαestimated using an eBioscience TNFα ELISA kit. While administration of1000 nM of Compound C or 2.5 nM of roflumilast alone, did not cause anappreciable response (<10% decrease of TNFα secretion), the combinationof the two compounds, when administered at the same concentration,resulted in a >30% decrease of TNFα secretion, i.e., greater than a3-fold increase in potency for the combination when compared with thepotency of Compound C or roflumilast when administered alone, therebyindicating a synergistic effect and therapeutic relevance of thiscombination in the treatment of inflammatory disorders, in particularairway disorders, psoriasis and RA.

LPS Induced TNFα in PBMC:

PBMC (peripheral blood mononuclear cells) from whole blood were isolatedby density gradient using Histopaque and incubated with the desiredconcentration of inhibitor for 15 minutes. LPS (1 μg/ml) was added andthen incubated for 24 hours. The supernatant was collected and TNFαestimated using an eBioscience TNFα ELISA kit. While administration of1000 nM of Compound C alone caused a 20% decrease of TNFα secretion,addition of 1.25 nM roflumilast to 1000 nM of Compound C resulted in a80% decrease of TNFα secretion, i.e., a 4-fold increase in potencycompared to the potency of Compound C alone, thereby indicating thesynergistic effect and therapeutic relevance of this combination in thetreatment of inflammatory disorders, in particular airway disorders,psoriasis and RA.

Lipopolysaccharide Induced Pulmonary Neutrophilia in Female Wistar Rats:

An exaggerated recruitment and subsequent activation of neutrophil islikely to be important for the development and course of severalinflammatory diseases in the airways and lungs, such as severe asthma,COPD, cystic fibrosis, and acute respiratory distress syndrome. Themechanisms by which neutrophils contribute to these diseases may involvethe release of proteolytic enzymes, such as neutrophil elastase, andfree oxygen radicals. When released, these agents can causebronchoconstriction, bronchial hyperreactivity, hyper-secretion,epithelial damage, and tissue remodelling in the airways.

After the quarantine period, fasted animals were randomized and dividedinto groups depending on their body weights. Test compound was preparedas a suspension in a vehicle consisting of 0.5% methylcellulose in whichTween 80 was used as a suspending agent. Compounds or vehicle wereadministered by oral gavage in a volume of 10 mL/kg. Animals wereanaesthetized with ketamine and LPS solution was administeredintratracheally 30 minutes after compound administration at a dose of 1mg/kg. Six hours after LPS instillation, animals were exsanguinatedunder anaesthesia, and the trachea was cannulated, and lungs werelavaged with 5-ml aliquots of heparinised PBS (1 unit/ml) four timesthrough the tracheal cannula (total volume 20 ml). Bronchioalveolar(BAL) fluid was stored at 2-8° C. until assayed for total cell anddifferential leukocyte count. BAL fluid was centrifuged (500×g for 10min) and the resulting cell pellet was resuspended in 0.5 ml ofheparinised saline. The total numbers of white blood cells weredetermined in BAL fluid or blood using a cell counter and were adjustedto 1×10⁶ cell/ml. Differential cell count was calculated manually. Onehundred microliters of the cell suspension was centrifuged usingcytospin 3 to prepare a cell smear. The cell smear was stained with ablood staining solution for differentiation and slides weremicroscopically observed to identify eosinophils according to theirmorphological characteristics. The number of each cell type among 300white blood cells in the cell smear was determined and expressed as apercentage of total cells. The number of eosinophil in BALF wascalculated.

Results:

The results are shown in FIGS. 7A, 7B, and 7C.

Effective Dose of Roflumilast:

Roflumilast demonstrated a dose dependent inhibition in neutrophilinfiltration compared to the control group at 0.3, 1, 3 and 10 mg/kg.Percent inhibitions were −7.89%, 43.46%, 68.02%, and 92.21% respectivelyand the 50% inhibition (ED₅₀) dose was 1.8 mg/kg.

Effective Dose of Compound C:

A dose dependent inhibition in neutrophil infiltration compared tocontrol group was observed at 0.1, 1, 3 and 10 mg/kg upon oraladministration of Compound C. Percent inhibitions were 14.15%, 57.76%,56.93%, and 81.55% respectively and the 50% inhibition (ED₅₀) dose was 1mg/kg.

Combination of Each ED₅₀ Dose of Roflumilast and Compound C:

Roflumilast or Compound C alone showed 36.18% and 36.56% inhibition ofneutrophil infiltration at doses of 1.8 and 1 mg/kg respectivelycompared to the control group. When roflumilast (1.8 mg/kg) was combinedwith Compound C (at a dose of 1 mg/kg), inhibition of neutrophilinfiltration increased to 78.20% compared to the control group animals

Acute Cigarette Smoke Induced Cell Infiltration in Male Balb/c Mice:

Animals were acclimatized for seven days prior to the start of theexperiment. Animals were randomly distributed to various groups based ontheir body weights. On day 1, mice were administered by test compound orvehicle by the oral route and after 30 mins test compound administrationanimals were placed in a whole body exposure box. The mice were exposedto the mainstream smoke of 6 cigarettes from day 1 to day 4. Exposure tothe smoke of each cigarette lasted for 10 min (cigarettes werecompletely burned in the first two minutes and followed by an air flowwith animal ventilator) followed by exposure for the next 20 min withfresh room air. After every second cigarette an additional break of 20min with exposure to fresh room air was conducted. Control animals wereexposed to the room air chamber. From day 1 to day 4 animals wereadministered test compound by the oral route. On day 5, 24 hours afterthe last cigarette smoke (CS) exposure animals were exsanguinated underanaesthesia, and the trachea was cannulated and the lungs were lavagedwith 0.5 ml aliquots of heparinised PBS (1 unit/ml) four times through atracheal cannula (total volume 2 ml). Bronchioalveolar (BAL) collectedwas stored at 2-8° C. until assayed for total cell and differentialleukocyte count. BAL fluid was centrifuged (500×g for 10 min) and theresulting cell pellet was resuspended in 0.5 ml of heparinised saline.The total number of white blood cells was determined in BAL fluid andblood using a blood cell counter and adjusted to 1×10⁶ cell/ml.Differential cell count was calculated manually. Forty microliters ofthe cell suspension was centrifuged using cytospin 3 to prepare a cellsmear. The cell smear was stained with a blood staining solution fordifferentiation and microscopically observed by identifying each cellaccording to its morphological characteristics. The number of each celltype among 300 white blood cells in the cell smear were determined andexpressed as a percentage, and the number of neutrophils and macrophagesin each BAL fluid were calculated.

Results:

The results are shown in FIG. 9.

Effective Dose of Roflumilast:

Roflumilast demonstrated a dose dependent inhibition in macrophageinfiltration compared to the control group at 1, 3 and 10 mg/kg. Percentinhibitions were 22.2%, 51.00%, and 69.11% respectively and the 50%inhibition (ED₅₀) dose was 3.5 mg/kg. Roflumilast demonstratedinhibition in neutrophil infiltration compared to the control group at1, 3 and 10 mg/kg. Percent inhibitions were 70.85%, 73.69%, and 83.01%respectively and the dose of 50% inhibition (ED₅₀) of neutrophilinfiltration was considered for combination study.

Effective Dose of Compound C:

A dose dependent inhibition in macrophage and neutrophil infiltrationcompared to the control group was observed at 1, 3 and 10 mg/kg uponoral administration of Compound C. Percent inhibitions of macrophageinfiltrations were 34.84%, 42.09%, and 61.77% respectively and the 50%inhibition (ED₅₀) dose was 4.4 mg/kg. Compound C demonstrated inhibitionin neutrophil infiltration compared to the control group at 1, 3 and 10mg/kg. Percent inhibitions were 29.06%, 62.38%, and 74.25% respectivelyand the dose of 50% inhibition (ED₅₀) of neutrophil infiltration was 2.1mg/kg.

Combination of each ED₅₀ Dose of Roflumilast & Compound C:

Roflumilast or Compound C alone showed 26.55% and 30.01% inhibition ofmacrophage infiltration at doses of 3.5 and 3 mg/kg respectivelycompared to the control group. When roflumilast (3.5 mg/kg) was combinedwith Compound C (at a dose of 3 mg/kg), inhibition of macrophageinfiltration increased to 99.89% compared to the control group animals.Roflumilast or Compound C alone showed 44.42% and 41.47% inhibition ofneutrophil infiltration at doses of 3.5 and 3 mg/kg respectivelycompared to the control group. Similarly, the combination of roflumilast(3.5 mg/kg) and Compound C (at a dose of 3 mg/kg) showed 88.34%inhibition of neutrophil infiltration compared to the control group.

Chronic Cigarette Smoke Induced Cell Infiltration in Male Balb/c Mice:

Animals were acclimatized for seven days prior to the start of theexperiment. Animals were randomly distributed to various groups based ontheir body weights. Mice were exposed to the mainstream smoke of 4cigarettes from day 1 to day 11. Exposure to the smoke of each cigarettelasted for 10 min (each cigarette was completely burned in the first twominutes, followed by an air flow with animal ventilator) and wereexposed for the next 20 min with fresh room air. After every secondcigarette an additional break of 20 min with exposure to fresh room airwas conducted. Control animals were exposed to the room air chamber.Test compound was administered by the oral route from day 6 to day 11before 30 mins whole body smoke exposure. On day 12, 24 hours after thelast cigarette smoke (CS) exposure animals were exsanguinated underanaesthesia, and the trachea was cannulated and the lungs were lavagedwith 0.5 ml aliquots of heparinised PBS (1 unit/ml) four times throughtracheal cannula (total volume 2 ml). Bronchioalveolar (BAL) collectedwas stored at 2-8° C. until assayed for total cell and differentialleukocyte count. BAL fluid was centrifuged (500×g for 10 min) and theresulting cell pellet was resuspended in 0.5 ml of heparinised saline.The total number of white blood cells was determined in BAL fluid andblood using a blood cell counter and adjusted to 1×10⁶ cell/ml.Differential cell count was calculated manually. Forty microliters ofthe cell suspension was centrifuged using cytospin 3 to prepare a cellsmear. The cell smear was stained with a blood staining solution fordifferentiation and microscopically observed by identifying each cellaccording to its morphological characteristics. The number of each celltype among 300 white blood cells in the cell smear was determined andexpressed as a percentage, and the number of neutrophils and macrophagesin each BAL fluid were calculated.

Results:

The results are shown in FIG. 11.

Combination of each ED₅₀ Dose of Roflumilast & Compound C:

Roflumilast or Compound C alone showed 6.30% and −13.30% inhibition ofmacrophage infiltration at dose of 1 and 1 mg/kg respectively comparedto the control group. When roflumilast (1 mg/kg) was combined withCompound C (at a dose of 1 mg/kg), inhibition of macrophage infiltrationincreased to 122.24% compared to the control group animals. Roflumilastor Compound C alone showed 34.60% and 4.08% inhibition of neutrophilinfiltration at dose of 1 mg/kg and 1 mg/kg respectively compared to thecontrol group. Similarly, the combination of roflumilast (1 mg/kg) andCompound C (at a dose of 1 mg/kg) showed 77.78% inhibition of neutrophilinfiltration compared to the control group.

Example 2 Combination Studies of a Dual PI3K Delta and Gamma Inhibitorand PDE-4 Inhibitor

This study was conducted using Compound A1 as the dual PI3K Delta andGamma inhibitor. Compound A1 exhibits an IC₅₀ value of <40 nM for boththe PI3K Delta and PI3K Gamma enzymes.

LPS Induced TNFα in MH-S (Mouse Alveolar Macrophage) Cells:

MH-S represents a mouse alveolar macrophage cell line that secretescopious amounts of TNFα upon induction by LPS. Cells were plated at150,000 cells per well. 10 nM of Compound A1 (final concentration) wasadded 15 minutes prior to the addition of roflumilast. LPS (1 μg/ml) wasadded and then incubated for 4 hours. The supernatant was collectedafter 20 hours and TNFα was estimated using an ELISA kit. Compound A1dose-dependently inhibited TNFα, a prominent cytokine involved in theprogression of COPD. Addition of 30 nM of Compound A1 to the PDE4inhibitor roflumilast resulted in a several-hundred fold reduction inIC₅₀ compared to the IC₅₀ of roflumilast alone, thereby indicating thesynergy and therapeutic relevance of this combination in airwaydisorders. Similarly, while administration of 15 nM of Compound A1 or130 nM of aprelimilast alone did not caused an appreciable decrease inTNFα (˜10%) secretion, the combination of the two compounds at the sameconcentrations resulted in a greater than 35% reduction, thus providingthe synergistic and therapeutic relevance of this combination in thetreatment of inflammatory disorders, in particular airway disorders,psoriasis and RA.

LPS Induced TNFα in THP-1 (Human Monocyte) Cells:

THP-1 represents a monocytic cell line that has elevated endogenous pAKTlevels and secretes copious amounts of TNFα upon induction by LPS. Cellswere plated at 150,000 cells per well. 10 nM of Compound A1 (finalconcentration) was added 15 minutes prior to the addition ofroflumilast. LPS (1 μg/ml) was added and then incubated for 4 hours. Thesupernatant was collected after 20 hours and TNFα was estimated using anELISA kit. Compound A1 dose-dependently inhibited TNFα, a prominentcytokine involved in the progression of COPD. Addition of 100 nMCompound A1 to the PDE4 inhibitor roflumilast resulted in aseveral-hundred fold reduction in IC₅₀ compared to the IC₅₀ ofroflumilast alone, thereby indicating the synergy and hence thetherapeutic relevance of this combination in the treatment of airwaydisorders.

Con A+PMA Induced IFNγ in Human Whole Blood (HWB):

Freshly collected HWB was diluted with media and incubated with thedesired concentration of inhibitor for 15 minutes. Cytokine release wasinduced with the addition of Concanavalin A (25 μg/ml)+Phorbol MyristateAcetate (50 ng/ml). The supernatant was collected after 20 hours andIFNγ was estimated using an ELISA kit. While administration of 10 nMCompound A1 alone did not affect IFNγ secretion, administration of thecombination of 10 nM Compound A1 and roflumilast resulted in a 3-foldreduction in IC₅₀ compared to the IC₅₀ of roflumilast alone, therebyindicating the therapeutic relevance of this combination in thetreatment of airway disorders, psoriasis and RA.

LPS Induced TNFα in Human Whole Blood (HWB):

Freshly collected HWB was diluted with media and incubated with thedesired concentration of inhibitor for 15 minutes. LPS (1 μg/ml) wasadded and then incubated for 24 hours. The supernatant was collected andTNFα estimated using an eBioscience TNFα ELISA kit. While administrationof 100 nM of Compound A1 alone did not affect TNFα secretion,administration of the combination of 100 nM Compound A1 and roflumilastresulted in a more that 10-fold reduction in IC₅₀ compared to the IC₅₀of roflumilast alone, thereby indicating the synergy as well thetherapeutic relevance of this combination in the treatment ofinflammatory disorders, in particular airway disorders, psoriasis andRA.

Con A+PMA Induced IFNγ in PBMC:

PBMC from whole blood were isolated by density gradient using Histopaqueand incubated with the desired concentration of inhibitor for 15minutes. Cytokine release was induced with the addition of ConcanavalinA (25 μg/ml)+Phorbol Myristate Acetate (50 ng/ml). Supernatant wascollected after 20 hours and IFNγ was estimated using an ELISA kit.While administration of 10 nM Compound A1 alone did not affect IFNγsecretion, addition of the combination of 10 nM Compound A1 androflumilast resulted in a 1.5-fold reduction in IC₅₀ compared to theIC₅₀ of roflumilast alone, thereby indicating the synergistic effect andthe therapeutic relevance of this combination in the treatment ofinflammatory disorders, in particular airway disorders, psoriasis andRA.

LPS Induced TNFα in PBMC:

PBMC from whole blood were isolated by density gradient using Histopaqueand incubated with the desired concentration of inhibitor for 15minutes. LPS (1 μg/ml) was added and then incubated for 24 hours. Thesupernatant was collected and TNFα estimated using an eBioscience TNFαELISA kit. While administration of 10 nM Compound A alone caused a 25%decrease of TNFα secretion, addition of the combination of 10 nMCompound A1 and roflumilast resulted in a 3-fold reduction in IC₅₀compared to the IC₅₀ of roflumilast alone, thereby indicating thetherapeutic relevance of this combination in the treatment ofinflammatory disorders, in particular airway disorders, psoriasis andRA.

Lipopolysaccharide Induced Pulmonary Neutrophilia in Female Wistar Rats:

The procedure for LPS induced pulmonary neutrophilia in female Wistarrats as described in Example 1 was performed with compound A1 androflumilast.

Results:

The results are shown in FIGS. 8A, 8B, and 8C.

Effective Dose of Roflumilast:

Roflumilast demonstrated a dose dependent inhibition in neutrophilinfiltration compared to the control group at 0.3, 1, 3 and 10 mg/kg.Percent inhibitions were −7.89%, 43.46%, 68.02%, and 92.21% respectivelyand the 50% inhibition (ED₅₀) dose was 1.8 mg/kg.

Effective Dose of Compound A1:

A dose dependent inhibition in neutrophil infiltration compared to thecontrol group was observed at 0.1, 1, and 10 mg/kg upon oraladministration of Compound A1. Percent inhibitions were 17.83%, 51.76%,and 70.21% respectively and the 50% inhibition (ED₅₀) dose was 1.3mg/kg.

Combination of each ED₅₀ Dose of Roflumilast and Compound A1:

Roflumilast or Compound A1 alone showed 36.18% and 43.02% inhibition ofneutrophil infiltration at dose of 1.8 and 1.3 mg/kg respectivelycompared to the control group. When roflumilast (1.8 mg/kg) was combinedwith Compound A1 (at a dose of 1.3 mg/kg), inhibition of neutrophilinfiltration increased to 79.20% compared to the control group animals

Acute Cigarette Smoke Induced Cell Infiltration in Male Balb/c Mice:

The procedure for acute cigarette smoke induced cell infiltration inmale Balb/c mice described in Example 1 was performed with Compound A1and roflumilast.

Results:

The results are shown in FIG. 10.

Effective Dose of Roflumilast:

Roflumilast demonstrated a dose dependent inhibition in macrophageinfiltration compared to the control group at 1, 3 and 10 mg/kg. Percentinhibitions were 22.2%, 51.00%, and 69.11% respectively and the 50%inhibition (ED₅₀) dose was 3.5 mg/kg. Roflumilast demonstratedinhibition in neutrophil infiltration compared to the control group at1, 3 and 10 mg/kg. Percent inhibitions were 70.85%, 73.69%, and 83.01%respectively and the dose of 50% inhibition (ED₅₀) of macrophageinfiltration was considered for combination study.

Effective Dose of Compound A1:

A dose dependent inhibition in macrophage and neutrophil infiltrationcompared to the control group was observed at 0.1, 0.3 and 1 mg/kg uponoral administration of Compound A1. Percent inhibitions of macrophageinfiltrations were 20.60%, 75.19%, and 93.11% respectively and the 50%inhibition (ED₅₀) dose was 0.20 mg/kg. Compound A1 demonstratedinhibition in neutrophil infiltration compared to the control group at0.1, 0.3 and 1 mg/kg. Percent inhibitions were 14.76%, 51.31%, and112.83% respectively and the dose of 50% inhibition (ED₅₀) of neutrophilinfiltration was 0.26 mg/kg.

Combination of Each ED₅₀ Dose of Roflumilast & Compound A1:

Roflumilast or Compound A1 alone showed 26.55% and 31.33% inhibition ofmacrophage infiltration at doses of 3.5 and 0.3 mg/kg respectivelycompared to the control group. When roflumilast (3.5 mg/kg) was combinedwith Compound A1 (at a dose of 0.25 mg/kg), inhibition of macrophageinfiltration increased to 65.52% compared to the control group animals.Roflumilast or Compound A1 alone showed 44.42% and 36.69% inhibition ofneutrophil infiltration at doses of 3.5 and 0.25 mg/kg respectivelycompared to the control group. Similarly, the combination of roflumilast(3.5 mg/kg) and Compound A1 (at a dose of 0.25 mg/kg) showed 79.14%inhibition of neutrophil infiltration compared to the control group.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as described above. It is intended that theappended claims define the scope of the invention and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

All publications, patents and patent applications cited in thisapplication are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference.

1. A method of treating an autoimmune, respiratory and/or inflammatorydisease or condition, the method comprising administering to a subjectin need thereof a therapeutically effective amount of (i) a PI3K Deltainhibitor or a dual PI3K Delta and Gamma inhibitor, and (ii) a PDE4inhibitor.
 2. The method according to claim 1, comprising administeringa PI3K Delta inhibitor.
 3. The method according to claim 1, comprisingadministering a dual PI3K Delta and Gamma inhibitor.
 4. The methodaccording to claim 1, wherein the PI3K Delta or dual PI3K Delta andGamma inhibitor is a compound of formula (I):

or a tautomer thereof, N-oxide thereof, pharmaceutically acceptableester thereof, prodrug thereof, or pharmaceutically acceptable saltthereof, wherein each occurrence of R is independently selected fromhydrogen, halogen, —OR^(a), CN, substituted or unsubstituted C₁₋₆ alkyl,substituted or unsubstituted C₂₋₆ alkenyl, substituted or unsubstitutedC₂₋₆ alkynyl, substituted or unsubstituted C₃₋₈ cycloalkyl, andsubstituted or unsubstituted heterocyclic group; R¹ and R² may be thesame or different and are independently selected from hydrogen, halogen,and substituted or unsubstituted C₁₋₆ alkyl, or both R¹ and R² directlybound to a common atom, may be joined to form an oxo group (═O) or asubstituted or unsubstituted saturated or unsaturated 3-10 member ring(including the carbon atom to which R¹ and R² are bound), which mayoptionally include one or more heteroatoms which may be the same ordifferent and are selected from 0, NR^(a) and S; Cy¹ is a monocyclicgroup selected from substituted or unsubstituted cycloalkyl, substitutedor unsubstituted heterocyclic group, substituted or unsubstituted aryland substituted or unsubstituted heteroaryl; Cy² is selected from asubstituted or unsubstituted heterocyclic group, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl; L₁ isabsent or selected from —(CR^(a)R^(b))_(q)—, —O—, —S(═O)_(q)—, —NR^(a)—or —C(═Y)—; each occurrence of R^(a) and R^(b) may be the same ordifferent and are independently selected from hydrogen, halogen,hydroxy, cyano, substituted or unsubstituted (C₁₋₆)alkyl, —NR^(c)R^(d)(wherein R^(c) and R^(d) are independently hydrogen, halogen, hydroxy,cyano, substituted or unsubstituted (C₁₋₆)alkyl, and (C₁₋₆)alkoxy) and—OR^(c) (wherein R^(c) is substituted or unsubstituted (C₁₋₆)alkyl) orwhen R^(a) and R^(b) are directly bound to a common atom, they may bejoined to form an oxo group (═O) or form a substituted or unsubstitutedsaturated or unsaturated 3-10 member ring (including the common atom towhich R^(a) and R^(b) are directly bound), which may optionally includeone or more heteroatoms which may be the same or different and areselected from O, NR^(d) (wherein R^(d) is hydrogen or substituted orunsubstituted (C₁₋₆)alkyl) or S; Y is selected from O, S, and NR^(a); nis 1, 2, 3 or 4; and q is 0, 1 or
 2. 5. The method according to claim 4,wherein the PI3K Delta or dual PI3K Delta and Gamma inhibitor is acompound of formula (II):

or a tautomer thereof, N-oxide thereof, pharmaceutically acceptableester thereof, prodrug thereof, or pharmaceutically acceptable saltthereof, wherein R, R¹, R², L₁, Cy¹ and Cy² are as described in claim 4.6. The method according to claim 4, wherein the PI3K Delta or dual PI3KDelta and Gamma inhibitor is a compound of formula (IA-I), (IA-II),(IA-HI) or (IA-IV):

or a tautomer thereof, N-oxide thereof, pharmaceutically acceptableester thereof, prodrug thereof, or pharmaceutically acceptable saltthereof, wherein each occurrence of X is independently selected from CR³or N; and each occurrence of R³ is independently selected from hydrogen,hydroxy, halogen, carboxyl, cyano, nitro, substituted or unsubstitutedalkyl, substituted or unsubstituted alkoxy, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted arylalkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkylalkyl, substituted or unsubstituted cycloalkenylalkylsubstituted or unsubstituted cycloalkenyl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted heterocyclic ring, substituted heterocyclylalkyl ring,substituted or unsubstituted guanidine,—COOR^(x), —C(O)R^(x),—C(S)R^(x), —C(O)NR^(x)R^(y), —C(O)ONR^(x)R^(y), —NR^(y)R^(z),—NR^(x)CONR^(y)R^(z),—N(R^(x))SOR^(y), —N(R^(x))SO₂R^(y),—(═N—N(R^(x))R^(y)), —NR^(x)C(O)OR^(y), —NR^(x)R^(y),—NR^(x)C(O)R^(y)—,—NR^(x)C(S)R^(y), —NR^(x)C(S)NR^(y)R^(z),—SONR^(x)R^(y)—, —SO₂NR^(x)R^(y)—, —OR^(x), —OR^(x)C(O)NR^(y)R^(z),—OR^(x)C(O)OR^(y)—, —OC(O)R^(x), —OC(O)NR^(x)R^(y),—R^(x)NR^(y)C(O)R^(z), —R^(x)OR^(y), —R^(x)C(O)OR^(y),—R^(x)C(O)NR^(y)R^(z), —R^(x)C(O)R^(x), —R^(x)OC(O)R^(y), —SR^(x),—SOR^(x), —SO₂R^(x), and —ONO₂, wherein R^(x), R^(y) and R^(z) in eachof the above groups can be hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted arylalkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkylalkyl, substituted or unsubstituted cycloalkenyl, substitutedor unsubstituted heteroaryl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted heterocyclic ring,substituted or unsubstituted heterocyclylalkyl ring, or substituted orunsubstituted amino, or any two of R^(x), R^(y) and R^(z) may be joinedto form a substituted or unsubstituted saturated or unsaturated 3-10membered ring, which may optionally include heteroatoms which may be thesame or different and are selected from O, NR^(F) (wherein R^(f) ishydrogen or substituted or unsubstituted alkyl) or S.
 7. The methodaccording to claim 1, wherein the PI3K Delta inhibitor is selected fromthe group consisting of:2-((6-amino-9H-purin-9-yl)methyl)-5-methyl-3-o-tolylquinazolin-4(3H)-one(IC87114),(S)-2-(1-((9H-purin-6-yl)amino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one(CAL-101),(S)-2-(1-(9H-purin-6-ylamino)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,and a pharmaceutically acceptable salt thereof.
 8. The method accordingto claim 1, wherein the dual PI3K Delta and Gamma inhibitor is selectedfrom the group consisting of:(S)-3-(1-((9H-purin-6-yl)amino)ethyl)-8-chloro-2-phenylisoquinolin-1(2H)-one(IPI-145),(+)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,(−)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,and a pharmaceutically acceptable salt thereof.
 9. The method accordingto claim 1, wherein the PDE-4 inhibitor is selected from the groupconsisting of enprofylline, theophylline, aminophylline, oxtriphylline,apremilast, roflumilast, cilomilast, tofimilast, pumafentrine,lirimilast, arofylline, atizorame, oglemilastum, D-4418, Bay-198004,BY343, CP-325,366, D-4396 (Sch-351591), AWD-12-281 (GW-842470), NCS-613,CDP-840, D-4418, PD-168787, T-440, T-2585, V 1 1294A, CI-1018, CDC-801,CDC-3052, D-22888, YM-58997, Z-15370,N-(3,5-dichloro-1-oxo-pyridin-4-yl)-4-difluoromethoxy-3-cyclopropylmethoxybenzamide,(−)-p-[(4aR*,10bS*)-9-ethoxy-1,2,3,4,4a,10b-hexahydro-8-methoxy-2-methylbenzo[s][1,6]-naphthyridin-6-yl]-N,N-diiso-propylbenzamid(R)-(+)-1-(4-bromobenzyl)-4-[(3-cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidon,3-(cyclopentyloxy-4-methoxyphenyl)-1-(4-N′-[N-2-cyano-S-methyl-isothioureido]benzyl)-2-pyrrolidone, cis[4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid],2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one,cis[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxy phenyl)cyclohexan-1-ol], (R)-(+)-ethyl[4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidin-2-yliden]acetate,(S)-(−)-ethyl[4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidin-2-yliden]acetate,9cyclopentyl-5,6-dihydro-7-ethyl-3-(2-thienyl)-9H-pyrazolo[3,4c]-1,2,4-triazolo[4,3a]pyridine and9-cyclopentyl-5,6-dihydro-7-ethyl-3-(tert-butyl)-9H-pyrazolo[3,4c]-1,2,4-triazolo[4,3 a]pyridine, and pharmaceutically acceptable saltsthereof.
 10. The method according to claim 1, wherein the PDE-4inhibitor is selected from the group consisting of theophylline,aminophylline, oxtriphylline, roflumilast, apremilast, andpharmaceutically acceptable salts thereof.
 11. The method according toclaim 1, wherein the therapeutically effective amount of the PI3K Deltaor dual PI3K Delta and Gamma inhibitor and the therapeutically effectiveamount of PDE4 inhibitor are administered simultaneously as a combinedformulation.
 12. The method according to claim 1, wherein thetherapeutically effective amount of PI3K Delta or dual PI3K Delta andGamma inhibitor and the therapeutically effective amount of PDE4inhibitor are administered sequentially.
 13. The method according toclaim 12, wherein the therapeutically effective amount of PDE-4inhibitor is administered before the therapeutically effective amount ofPI3K Delta or Dual PI3K Delta and Gamma inhibitor.
 14. The methodaccording to claim 1, wherein the therapeutically effective amount ofPI3K Delta or dual PI3K Delta and Gamma inhibitor is administered twicedaily to once every three weeks, and the therapeutically effectiveamount of the PDE4 inhibitor is administered twice daily to once everythree weeks.
 15. The method according to claim 1, wherein theautoimmune, respiratory and/or inflammatory disease or condition isselected from the group consisting of asthma, chronic obstructivepulmonary disease, rheumatoid arthritis, inflammatory bowel disease,glomerulonephritis, neuroinflammatory diseases, multiple sclerosis,uveitis, psoriasis, arthritis, vasculitis, dermatitis, osteoarthritis,inflammatory muscle disease, allergic rhinitis, vaginitis, interstitialcystitis, scleroderma, osteoporosis, eczema, allogeneic or xenogeneictransplantation (organ, bone marrow, stem cells and other cells andtissues) graft rejection, graft-versus-host disease, lupuserythematosus, inflammatory disease, type I diabetes, pulmonaryfibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis, myastheniagravis, autoimmune hemolytic anemia, cystic fibrosis, chronic relapsinghepatitis, primary biliary cirrhosis, allergic conjunctivitis, atopicdermatitis, and combinations thereof.
 16. The method according to claim1, wherein the autoimmune, respiratory and/or inflammatory disease orcondition is selected from the group consisting of asthma, allergicrhinitis, non-allergic rhinitis, rheumatoid arthritis, chronicobstructive pulmonary disease, and atopic dermatitis.
 17. The methodaccording to claim 1, wherein the PI3K Delta or dual PI3K Delta andGamma inhibitor and at the PDE4 inhibitor are each administered in anamount ranging from about 0.01 mg to about 1000 mg.
 18. The method ofclaim 1, wherein the PI3K Delta or dual PI3K Delta and Gamma inhibitorand the PDE4 inhibitor are administered at a ratio of about 1:100 toabout 100:1 by weight.
 19. A pharmaceutical composition comprising (i) aPI3K Delta or dual PI3K Delta and Gamma inhibitor, or a pharmaceuticallyacceptable salt thereof, (ii) a PDE4 inhibitor, and (iii) optionally, apharmaceutically acceptable carrier, glidant, diluent, or excipient.20-29. (canceled)
 30. A kit for treating an autoimmune, respiratory orinflammatory disease or condition, the kit comprising: (i) a PI3K Deltaor PI3K Delta and Gamma inhibitor, and (ii) a PDE4 inhibitor, either ina single pharmaceutical composition or in separate pharmaceuticalcompositions, (ii) optionally, instructions for treating the autoimmune,respiratory or inflammatory disease or condition with the PI3K Delta orPI3K Delta and Gamma inhibitor, and PDE4 inhibitor, and (iii)optionally, a container for placing the pharmaceutical composition orpharmaceutical compositions. 31-34. (canceled)